Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy

Abstract

Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

Fig. 1

Comparison of epifluorescence versus total internal reflection fluorescence microscopy. The thin layer of illumination is an evanescent field produced by an excitation light beam in a glass cover slip that is incident at a high angle upon the solid-solution interface at which the cells adhere. Thus, an evanescent wave arises on the cell–substrate interface and penetrates a small distance (∼150 nm) into the cells.

Fig. 2

Arrangement for objective lens-type TIRF in an inverted microscope. Laser illumination through a side port requires a dichroic mirror cube facing the side. At the P1 position, the laser beam is focused to lead the critical angle propagation into the coverslip (total internal reflection). Moving the lens L transversely changes the angle of incidence, thus the laser beam moves from P1 (TIRF) position to P2 position (epifluorescence). This system allows to switch between both mode of illumination (TIRF and epifluorescence).

Fig. 3

Epifluorescence versus objective lens-type total internal reflection fluorescence. Images were captured with Olympus 1.45 NA 100× objective lens and an argon ion laser source of wavelength 488 nm, using the side-port configuration depicted in Fig. 2. (A and B) PC12 cells containing secretory vesicle marker neuropeptide Y-Venus (NPY-Venus). (C and D) PC12 cells containing secretory vesicle marker rabphilin-mRFP. The images were recorded by a cooled monochrome CCD camera (Imago, Till Photonics) in A and B, and recorded by EM-CCD camera (C9100-02, Hamamatsu Photonics) in C and D.

Fig. 4

Analysis of secretion by TIRF microscopy. (A) Evanescent wave image of NPY-Venus fluorescence in a live MIN6 pancreatic β cell. The scale bar represents 5 μm. (B, top) Sequential images of a single vesicle observed after high [K⁺] stimulation. The third image shows a diffuse cloud of the NPY-Venus fluorescence, and the final image shows an abrupt disappearance of the fluorescent spot. (B and C, bottom) Time course of the fluorescence changes measured in the small circles enclosing fluorescent spots (filled symbols) and for concentric annuli around the circles (open symbols) of two different vesicles. The ordinate is given in arbitrary units of brightness. (C, top) Sequential images of a single vesicle observed after stimulation with 50 mM KCl without showing any diffuse cloud of the NPY-Venus fluorescence. The third image does not show any cloud of the dye, whereas the final image shows an abrupt disappearance. These events typically exhibited a much slower time course (approximately 3 s to reach peak fluorescence) than those showing in B and reflects the approach of vesicles to the plasma membrane without exocytosis (i.e. vesicle movement or retrieval). The scale bars represents 1 μm. (D) Effect of glucose on exocytosis as reported with NPY-Venus in pancreatic β cells. The NPY-Venus spots shown in B was counted manually as secretion events every 60 s and plotted against time. Stimulation with high glucose concentrations (30 mM) caused a marked increase in the number of secretion.